Bacillus anthracis, the causative agent of anthrax, is one of the six CDC category A agents considered to be those which would cause the most adverse public health impact if used in a biological attack. Highly virulent forms of B. anthracis secrete two toxins, lethal toxin (LeTx) and edema toxin (EdTx), which are thought to be primarily responsible for the major symptoms and death associated with anthrax. Entry of both types of toxin into cells is mediated by the bacterial protective antigen (PA) subunit. In an effort to understand how these toxins enter cells we have identified two distinct cellular receptors for PA, Anthrax Toxin Receptor/Tumor Endothelial Marker-8 (ATR/TEM8) and Capillary Morphogenesis Protein-2 (CMG2). We have also developed soluble receptor decoys as candidate antitoxins for the treatment of anthrax. The research described in this proposal will build upon these landmark findings to further our understanding of anthrax toxin entry. The first aim will investigate the role played by the receptors in trafficking toxin to distinct sites in the cell that are associated with marked differences in toxin stability. The second aim will seek to develop a cell-free system that will allow the dissection of the molecular mechanism of anthrax toxin translocation. The third aim will investigate whether anthrax toxin, like other bacterial toxins, can be targeted to other types of cell surface receptors (surrogate receptors). This information will provide important insights into the critical parameters required for toxin entry. The fourth aim will seek to establish whether the DNI-class of anthrax antitoxins can block toxin entry that is mediated by surrogate receptors. Together, this body of work will contribute significantly to our understanding of the cell biology of anthrax toxin entry. This information in turn may reveal new targets for therapeutic intervention in the treatment of disease, and may pre-empt the actions of bioterrorists who seek to target PA to surrogate cell surface receptors in an effort to evade anti-PA antibody and soluble receptor decoy-based antitoxins.